Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The overall goal of this procedure is to analyze the migratory behavior of cells within the three dimensional environment. First, embed the cells of interest into a three dimensional collagen lattice. Then record the movement of the cells by time lapse video microscopy next, delineate the migration parameters by computer assisted cell tracking and analyze the cell tracking data by using a spreadsheet software application.

Ultimately, this method can be used to investigate which factors and signal transduction pathways regulate the migration of cancer cells or normal cells like leukocytes, lymphocytes, and stem cells In the context of cancer. So knowledge of cell migration, modulating factors and associated signal transection pathways might be helpful for the development of novel therapeutic strategies to impair tumor cell migration and metastatic spreading. For the migration chambers rapidly brush two layers of melted paraffin wax, petroleum jelly mix to the middle of a glass slide, add a cover, slip and seal with two layers of the paraffin wax, petroleum jelly mix.

Then place the migration chambers in an upright position in a rack to 1.5 milliliter reaction tubes. Add 20 microliters of the cell suspension for the collagen suspension. Combine 50 microliters of 10 XMEM and 27 microliters of 7.5%Sodium bicarbonate solution mix thoroughly.

Then add 375 microliters of liquid collagen suspension and mix thoroughly for a light purple color indicating a pH of 7.5. Now add 100 microliters of the final collagen suspension to 50 microliters of the cell suspension and mix thoroughly while noting the initiation of collagen fiber.Polymerization. At pH 7.5, transfer the mix to the migration chambers.

Gently tap the migration chamber to equally distribute the collagen suspension cell mix on the bottom of the migration chain chamber. Place the migration chambers in an upright position in rack and incubate for 30 minutes. When the polymerized collagen lattice is slightly turbid, but still a light purple color, fill the migration chambers with the appropriate medium and seal the migration chamber with paraffin wax.

Petroleum jelly mix. Adjust the microscope stage heater to 37 degrees Celsius using a 10 x magnification on the migration chamber. Focus approximately 50 cells or more in the field of view.

Record the cell migration in time lapse mode using a multi-camera video surveillance software application. Save the cell migration movies in an appropriate file format. Link the cell migration movie files to a database containing supporting information such as cell type and experimental conditions.

Next, analyze the cell migration movies using an appropriate cell tracking software application for an unbiased analysis. Randomly select the cells for analysis without the knowledge of whether cells are migratory active in total, determine about 60 XY coordinates per cell in a typical cell migration experiment. In case of cell division, randomly choose one daughter cell to follow in tracking.

Analyze cell tracking data by using an appropriate software application like a spreadsheet program to calculate the total distance that a cell has migrated. Multiply the sum of the numerical pixel values by a correction factor for conversion into microns. Define the cells that have migrated less than the 25 microns threshold as non-moving cells, and replace the numerical values with a comma.

Next, calculate the locomotory activity as a percentage of cells of the tracked cell population that have moved between two time points. Also, calculate the time of active movement as a percentage of the total time a cell has migrated in relation to the timeframe of the observation period. Then calculate the statistical significance using the man Whitney U test and display the cell migration data.

A typical cell tracking data file resembles a table where each column stands for one tracked cell. The rows contain the positional information on cell movement. Over time, the locomotive behavior of cells can be characterized by the mean locomotor activity of the analyzed cell population and also the time of active movement.

This example shows the migratory activities of 120 cells from four independent experiments. Each diamond indicates that a cell has moved between at least two time points. The gray lines differentiate the moving and non-moving cells.

The parameter of locomotive activity can be displayed as an XY diagram or a box plot. To visualize the dynamics of the analyzed cell population, a histogram effectively shows the time active of those same cells. This analysis of the migratory behavior of MDA HER2 and M-D-A-N-E-O breast cancer cell lines indicates that both cell lines exhibited similar spontaneous locomotor activity.

The parameter time active revealed a slightly higher number of spontaneously moving M-D-A-H-E-R cells as compared to M-D-A-N-O breast cancer cells. Stimulation with 100 nanograms per milliliter of EGF resulted in a significantly increased migratory activity of MDA HER2 as measured by the number of moving cells and time of active movement. Interestingly, M-D-A-N-E-O cells migrated in response to EGF stimulation and displayed a shifted time active pattern treatment with phospholipase c gamma one inhibitor uc 3 1 22 reduced both the spontaneous and EGF induced migration of both M-D-E-E-R two and MDAN.

EEO cells. Analysis of the time active parameter revealed that treatment of MDAN EEO cells with two micromolar of U 7 3 1 2 2 resulted in a markedly decreased locomotor activity, primarily attributed to an increased amount of non-moving cells. Once mastered migration of lymphocytes and leukocytes into a three dimensional collagen letters and ment can be recorded in about two hours where tumor cells are recorded overnight.